Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) catalyzes the reduction of atmospheric CO2 during photosynthesis. This process is not efficient and methods to improve the efficiency of CO2 reduction are needed. Methods which improve the efficiency of CO2 reduction will increase plant growth rates, which will be useful to a variety of industries.
A great deal is known about the quaternary structure, catalytic mechanism, active site residues, in vivo regulatory mechanisms, and gene expression for this abundant enzyme, (see, for example, Andrews et al., “Rubisco: Structure, Mechanisms, and Prospects for Improvement,” in Hatch et al. (eds), The Biochemistry of Plants, vol, 10, pp. 131-218. Academic Press, York (1987); Dean et al., “Structure, evolution, and regulation of rbcS genes in higher plants,” Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 415-439 (1989); and Mullet, “Chloroplast development and gene expression,” Annu. Rev. Plant. Physiol. Plant Mol. Biol. 39: 475-502 (1988)). Higher plant Rubisco is a hexadecameric protein composed of eight chloroplast-encoded large subunits (referred to herein as “LS”) and eight nuclear-encoded small subunits (referred to herein as “SS”). Synthesis of the LS is accompanied by post-translational processing of the N-terminal domain (Houtz et al., “Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase,” Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989); and Mulligan et al., “Reaction-intermediate analogue binding by ribulose bisphosphate carboxylase/oxygenase causes specific changes in proteolytic sensitivity: The amino-terminal residue of the large subunit is acetylated proline,” Proc. Natl. Acad. Sci. USA 85:1513-1517 (1988)). The N-terminal Met-1 and Ser-2 are removed and Pro-3 acetylated. Additionally, the LS of Rubisco from tobacco, muskmelon, pea, and several other species is post-translationally modified by trimethylation of the ε-amine of Lys-14 (Houtz et al., “Posttranslational modifications in the amino-terminal region of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from several plant species,” Plant Physiol. 98:1170-1174 (1992); Houtz et al., “Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase,” Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989)). The enzyme responsible for this latter modification is a highly specific chloroplast-localized S-adenosylmethionine (AdoMet):protein (lys) εN-methyltransferase (protein methylase III, Rubisco LSMT, EC 2.1.1.43) (Houtz et al., “Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase,” Proc. Natl. Acad. Sci. USA 86:1855-1859 (1989)).
Rubisco LSMT has been affinity purified-8000-fold from pea chloroplasts and identified as a monomeric protein with a molecular mass of ˜57 kDa (Wang et al., “Affinity Purification of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit εN-Methyltransferase,” Protein Expression and Purification 6:528-536 (1995)). Recently, Rubisco LSMT cDNAs have been cloned and sequenced from pea and tobacco (Klein et al., “Cloning and developmental expression of pea ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit N-methyltransferase,” Plant Molecular Biol. 27:249-261 (1995); Ying et a I., “Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit εN-methyltransferase gene in tobacco,” Plant Molecular Biology 32:663-671 (1996)). The deduced amino acid sequences of tobacco Rubisco LSMT has 64.5% identity and 75.3% similarity with the sequence of pea Rubisco LSMT, and both proteins contain several copies of a conserved imperfect leucine-rich repeat motifs (Ying et al., “Organization and characterization of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit εN-methyltransferase gene in tobacco,” Plant Molecular Biology 32:663-671 (1996)).
Kaplan et al., in U.S. Pat. No. 6,320,101, addressed the need for increased efficiency of Rubisco CO2 reduction by over-expressing a gene in a cell. However, the efficiency of CO2 reduction in this method is not optimal and there is a need for additional methods of altering Rubisco carboxylase activity. There is a need for improved methods of targeting molecules to Rubisco.